Katarzyna H. Kucharzyk, Jayda E. Meisel, Fadime Kara-Murdoch, Robert W. Murdoch, Steven A. Higgins, Simon Vainberg, Craig M. Bartling, Larry Mullins, Paul B. Hatzinger, Frank E. Löffler
At groundwater sites contaminated with chlorinated ethenes, fermentable substrates are often added to promote reductive dehalogenation by indigenous or augmented microorganisms. Contemporary bioremediation performance monitoring relies on nucleic acid biomarkers of key organohalide-respiring bacteria, such as Dehalococcoides mccartyi (Dhc). Metagenome sequencing of the commercial, Dhc-containing consortium, SDC-9, identified 12 reductive dehalogenase (RDase) genes, including pceA (two copies), vcrA, and tceA, and allowed for specific detection and quantification of RDase peptides using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Shotgun (i.e., untargeted) proteomics applied to the SDC-9 consortium grown with tetrachloroethene (PCE) and lactate identified 143 RDase peptides, and 36 distinct peptides that covered greater than 99% of the protein-coding sequences of the PceA, TceA, and VcrA RDases. Quantification of RDase peptides using multiple reaction monitoring (MRM) assays with 13C-/15N-labeled peptides determined 1.8 × 103 TceA and 1.2 × 102 VcrA RDase molecules per Dhc cell. The MRM mass spectrometry approach allowed for sensitive detection and accurate quantification of relevant Dhc RDases and has potential utility in bioremediation monitoring regimes.
Kucharzyk KH, Meisel JE, Kara-Murdoch F, Murdoch RW, Higgins SA, Vainberg S, Bartling CM, Mullins L, Hatzinger PB, Loffler FE. 2020. Metagenome-guided proteomic quantification of reductive dehalogenases in the Dehalococcoides mccartyi-containing consortium SDC-9. Journal of Proteome Research 19:1812-1823.